ES2309985T3 - HEMATOPOYETIC FACILITATING CELLS AND THEIR USES. - Google Patents

Abstract

THE PRESENT INVENTION REFERS TO HAMATOPOYETIC FACILITATING CELLS OF MAMIFEROS (FC). IN PARTICULAR IT REFERS TO THE ISOLATION, CHARACTERIZATION AND USES OF THE FC. THE FC OF THIS INVENTION MAY BE DIFFERENTIATED FROM OTHER CELLS OF THE OSEA MEDULA KNOWN BY ITS MORPHOLOGY, THE PHENOTYPE OF THE CELL SURFACE AND THE IN VIVO FUNCTION. IT HAS BEEN DISCOVERED THAT CELLS WITH PURIFIED HEMATOPOYETIC SIZE ONLY ONLY OR THE OSEA MEDULA CELLS WITHOUT FC ARE NOT EASILY INTEGRATED IN A CONTAINER. WHEN THEY ARE ADMINISTERED TOGETHER WITH OTHER OSEA MEDULA CELLS, ESPECIALLY CELLS WITH A HEMATOPOIETIC SIZE IN A CONTAINER, THE FC INCREASES ITS CAPACITY TO BE INJECTED, WITHOUT ADVERSE ADVERSE BIOLOGICAL ACTIVITIES. IN FACT, THE CAPACITY OF THE FCS TO INCREASE THE CAPACITY THAT THE CELLS OF THE MEDULA OSEA TO BE INTEGRATED TO ESTABLISH A LINFOHEMATOPOYETIC CHEMERISM WITHOUT PRODUCING AN INJERT AGAINST A HOSPITAL DISEASE ALSO INJURIES THE FOLLOWING PERMITATION OF THE PERMIT DONATOR CELLS, FABRICS AND ORGANS. THEREFORE, THE FC MAY HAVE A WIDE RANGE OF APPLICATIONS, INCLUDED, BUT NOT RESTRICTED TO, THE HEMATOPOYETIC RECONSTITUTION THROUGH A TRANSPLANT OF THE OSEA MEDULA FOR THE TREATMENT OF CANCERES, ANEMIAS, SELF-IMMUNITIES, IMMUNODECTIVES AND ASSOCIATION FACILITATE THE TRANSPLANT OF SOLID ORGANS, FABRICS AND CELLS.

Description

Hematopoietic facilitating cells and their applications.

1. Introduction

The present invention relates to mammalian hematopoietic facilitator (CF) cells. In particular, it relates to the isolation, characterization and use of CF. The CFs of the present invention can be distinguished from all other known bone marrow cells by their morphology, cell surface phenotype and in vivo function . It has now been established that purified hematopoietic stem cells alone or bone marrow cells depleted of CF is not readily grafted to a receptor. When co-administered with other bone marrow cells, especially hematopoietic stem cells in a recipient, CFs improve their grafting, with no apparent adverse biological activities. In fact, the ability of CFs to improve bone marrow cell grafting in establishing lymphohematopoietic chimerism without producing graft versus host diseases also induces the tolerance of specific donors to allow permanent donor acceptance of cells, tissues and organs. . Therefore, CFs can have a wide range of applications, including, but not limited to, the hematopoietic reconstitution of bone marrow transplantation for the treatment of cancers, anemias, autoimmunity, immunodeficiency, viral infections and metabolic disorders, as well as facilitation of solid organs, tissues and cell transplantation.

2. Background of the invention

One of the main objectives in the Solid organ transplant is the organ grafting of the donor, without graft rejection due to a generated immune response by the receiver, while preserving the immunocompetence of the receptor against other antigens strangers. Typically, nonspecific immunosuppressive agents, such as cyclosporine, methotrexate, steroids and FK506 are used to prevent guest rejection responses. They must be administered daily and, if interrupted, usually graft rejection results. However agents nonspecific immunosuppressants work by suppressing all aspects of the immune response, which greatly increases the susceptibility of the recipient to infections and diseases, including cancer. Moreover, despite the use of immunosuppressive agents, graft rejection remains a important source of morbidity and mortality in the transplant of human organs Only 50% of heart transplants 5 years survive and 20% of kidney transplants survive 10 years. (See Powles, 1980, Lancet, p. 327; Ramsay, 1982, New Engl. J. Med., P. 392). Most human transplants They fail within 10 years without permanent acceptance. For the therefore, it would be a breakthrough if tolerance could be induced in the receiver

The only known clinical condition in which complete systemic tolerance of specific donor transplantation occurs is when chimerism occurs through bone marrow transplantation. (See Qin et al ., 1989, J. Exp. Med. 169: 779; Sykes et al ., 1988, Immunol. Today 9:23; Sharabi et al ., 1989, J. Exp. Med. 169: 493) . This has been achieved in neonatals and in adult animal models, as well as in humans due to the total lymphoid irradiation of a recipient followed by bone marrow transplanted with donor cells. The bone marrow transplant success rate is, in part, dependent on the increased ability to closely combine the histocompatibility complex (MHC) of donor cells with those of the cell receptor. MHC is a complex gene that encodes a wide variety of individually unique glycoproteins expressed individually on the donor and recipient's cell surface, which are the main targets of the transplant rejection immune response. In humans, the MHC is called HLA. When the HLA identity is achieved by adapting a patient with a family member such as a sibling, the probability of a successful outcome is relatively high, despite graft- versus- host disease (GVHD) is not yet completely removed. However, when allogeneic bone marrow transplantation is performed between two individuals not combined with MHC of the same species, common complications involve graft failure, poor immunocompetence and a high incidence of GVHD.

GVHD is a potentially lethal complication in bone marrow transplantation, which occurs in approximately 35-50% of untreated HLA-identical marrow graft recipients (Martin et al ., 1985, Blood 66: 664) and up to 80% of bone marrow recipients not combined with HLA. Unfortunately, only 30% of patients in general have an HLA-identical properly combined with a member of the donor family, and therefore, most patients are either excluded from being considered for bone marrow transplantation, or if they are transplanted they must tolerate a high risk of GVHD. Results of GVHD capacity of immunocompetent mature immune cells (mainly T cells, but some B cells and natural killer cells) in the donor graft to recognize host tissue antigens as foreign and invoke an adverse immune reaction. Although mixed allogeneic reconstitution, in which a mixture of bone marrow donors and recipients is transplanted, results in improved immunocompetence and increased resistance to GVHD, grafting success is not yet systematically achieved and GVHD still occurs at often.

Recent studies in bone marrow transplantation suggest that the main cause of GVHD is T cells, as well as the removal of T cells from the donor cell preparation was associated with a reduction in the incidence of GVHD. (Vallera et al ., 1989, of Transplants, 47: 751; Rayfield, 1984, Eur. J. Immunol., P. 308; Vallera, 1982, J. Immunol., 128: 871; Martin and Korngold, 1978, J Exp. Med., P 1687; Prentice, 1984, Lancet p. 472). After T cells were implicated to be the predominant mediators of GVHD in animal models, aggressive protocols for cell depletion (TCD) of human bone marrow donors were instituted. Although the incidence of GVHD was drastically reduced, TCD was accompanied by a significant increase in graft failure, indicating that T cells can also play a positive role in bone marrow grafting. (Soderling, J. Immunol., 1985, 135: 941; Vallera, 1982, of Transplants. 33: 243; Pierce, 1989, of Transplants., P. 289). The increase in graft failure in human recipients ranges from 5-70% of the total number of patients and is related to the degree of MHC disparity between the donor and the recipient (Blazar, 1987, UCLA Symp., P. 382; FILIPOVICH, 1987, Transplant., P. 62; Martin et al ., 1985, Blood 66: 664; Martin et al ., 1988, Adv. Immunol. 40: 379). Patients with a failed graft usually do not die although a second bone marrow transplant is performed. Consequently, most transplant institutions in the United States have abandoned the bone marrow donor TCD and, therefore, must tolerate a high level of GVHD that leads to significant morbidity and mortality. Thus, the application of bone marrow transplantation as a form of treatment is limited only to places where the potential of GVHD is clearly exceeded by the potential benefit.

The implication that T cells could participate in both harmful reactions GVHD and in the useful grafting facilitation was an enigma that existed during Long time in the scientific community. Investigators they began to look for the possible existence of a component of the bone marrow that could facilitate bone grafting bone, but was removed during TCD. Identification and purification of this facilitator component could potentially allow the transplant protocol project to prevent selectively GVHD, by preserving the cells that could improve grafting

Although most researchers speculated that the facilitator component was a hematopoietic cell other than that of hematopoietic stem cells, such a component had never been identified or characterized before the present invention In fact, all the evidence pointed to the participation of some type of T cells. There was a desperate need for accurate knowledge of the identity of this component that could facilitate cell grafting Hematopoietic mother in a receptor without producing GVHD.

3. Summary of the invention

The present invention satisfies the above described need identified for some time. The present invention relates to mammalian hematopoietic CF methods of cell isolation, and methods of using cells to facilitate reconstitution of damaged autologous or destroyed, syngeneic, allogeneic hematopoietic system or xenogeneic with stem cells, as well as to induce tolerance in specific donors for the transplantation of cells, tissues and solid donor organs.

The invention is based, in part, on the discovery by the Applicant that the bone marrow murine contains a population of cells that indicate a phenotype of THY1 +, MHC Class II + (only darken at intermediate levels of expression), CD45 +, CD45R +, CD8 +, CD3 + and? /? TCR - which are able to facilitate allogeneic grafting of bone marrow from donors in a recipient. Both procedures of negative selection that involves the removal of these cells and the positive selection methods that involve the incorporation of these cells in a highly purified or partially purified confirm that they have facilitating activities of grafting and distinguish themselves from the T cells responsible for the GVHD Morphologically, purified CFs are different from all other types of hematopoietic cells, including lymphocytes. Moreover, these cells work in a way MHC-specific in which the optimal grafting of bone marrow cells are achieved if they have the same MHC haplotype of the CF. The CFs of the invention can also mediate xenogeneic grafting in the bone marrow through the species barriers in the establishment of chimerism mixed lymphohematopoietic.

The invention is described by way of examples. in which mouse, rat, baboon and human CFs are isolated, and its cell surface phenotype is characterized. CF isolated are used to successfully establish grafting of bone marrow cells from donors without the manifestation of GVHD. In addition, bone marrow cell donors deplete cells GVHD producers, especially T cells, with retention of CFs also produce grafting and mixed chimerism, which It makes the recipient immunologically tolerant to donors. A wide variety of uses for CFs are covered within the invention described in this section, including, but not limited to, transplants, and cancer treatment, disorders metabolic, immunodeficiency, autoimmunity, diabetes, hemoglobinopathies, hepatitis, AIDS and aging.

The present invention provides the methods of purification of CF from bone marrow or other physiological sources  of hematopoietic cells. CFs are purified by separation based on the presence or absence of specific markers.

By using the ability of CFs to facilitate bone marrow or stem cell grafting purified and therefore establish an immune system chimeric hematopoietic, the present invention provides methods of transplantation that confer the transplant tolerance of donor-specific and eliminates the need for agents  non-specific immunosuppressants

It is an object of the present invention provide a cellular composition comprising CF purified or partially purified hematopoietic.

It is a new object of this invention to provide a cellular composition comprising Purified or partially purified hematopoietic CF and cells purified or partially purified hematopoietic mother that are MHC-specific for FC.

Another object of the present invention is provide a cellular composition comprising CF purified or partially purified hematopoietic cells hematopoietic mother who are MHC-specific at FC, and one or more new components of hematopoietic cells that they are MHC - specific to CF.

It is a new object of this invention to provide a cellular composition comprising Hematopoietic CF and hematopoietic stem cells in which only the T cells responsible for GVHD have been especially and selectively removed.

It is a further object of the present invention provide hematopoietic CF purification methods of physiological sources of hematopoietic cells.

It is another object of the present invention provide methods to establish a mixed allogeneic system, mixed xenogeneic, completely allogeneic, and completely immune Xenogenic chimeric in a receptor.

It is another object of the present invention provide methods of transplantation of a physiological component of the  donor in a recipient that allows transplant tolerance donor-specific.

It is another object of the present invention provide methods of treatment of a variety of diseases and bone marrow transplant disorders that involve CF.

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3.1. Definitions

As used in this document, "receptor" means any mammal, including beings humans.

As used in this document, "donor" means every mammal, including beings humans.

As used in this document, except in the case that traditionally explicitly understood referred to, "MHC-specific" cells involve cells whose major histocompatibility complex does not prevent the facilitating cell from facilitating grafting, if major histocompatibility complex cells is actually identical to the facilitating cell or, in the case of a cell universal facilitator is simply not a barrier to grafting

As used in this document, "purified" means any enrichment or increase in the concentration of cells specific to their natural state, between They isolate these cells.

As used in this document, "substantially destructive" means the means to destroy all or almost the entire immune system of receivers

As used in this document, "lethal radiation" is understood as the means to destroy substantially the recipient's immune system with radiation.

As used in this document, "immunosuppressants" is understood as the means to suppress the functions of the recipient's immune system, including the suppression of the propensity to attack cells recognized strange (ie rejection of a graft).

As used in this document, "cytoreducido" means to destroy a part of the cells of the recipient's immune system in order to make physical space for Immune cells administered.

As used in this document, "the donor physiological component "means any part or combination of donor body parts, including organs, tissues and cells.

As used in this document, "chimera" is understood as receptor cells and al cells Less a donor.

As used in this document, "syngeneic" means the origin of the donor in which the Donor is genetically identical to the recipient.

As used in this document, "allogeneic" means the origin of the donor in which the Donor is from the same species of the recipient.

As used in this document, "xenogeneic" means the origin of the donor in which the Donor is of a different species from that of the recipient.

As used in this document, "mixed chimeric immune system" is understood as the system immune system that comprises about 0.5% to 99% of allogeneic or xenogenic cells and the remaining percentage of syngeneic cells.

As used in this document, "chimeric immune system of completely allogeneic cells" it is understood by the immune system of the receptor created to through the administration of both allogeneic cells and syngeneic and practically comprising 100% allogeneic cells, but in which some residual synergic cells, which provide a limited number of immune lineage cells, May exist.

As used in this document, "completely xenogenic chimeric immune system" is understood by the immune system of the receptor created through the administration of both allogeneic and syngeneic cells and that It comprises practically 100% allogeneic cells, but in which Residual synergic cells, which provide a number Limited immune cell lineages may exist.

4. Brief description of the drawings

Fig. 1 CF Electron Micrograph purified.

5. Detailed description of the invention

The present invention relates to CF mammalian hematopoietic, to the methods of isolating and characterizing the CF, and the methods of using it.

While the initial studies on negative selection were directed to the view that the facilitating hematopoietic cells were CD8-, subsequent studies described herein have led to the conclusion that the appropriate phenotype of CFs includes CD8 +. The experimental data contained in this document that lead to the early conclusion regarding CD8- is retained for informational purposes only (see Section 6, infra ), as the facilitating cell is now conclusively proven to be CD8 + by positive selection studies (see section 7, infra ). These seemingly contradictory results are probably due to incomplete removal of cells expressing CD8 at low density during antibody treatment, more complete in the negative selection method used in section 6. In flow cytometry studies using anti-antibody -CD8 monoclonal for positive selection, a small population of CD8 + cells is identified, which exhibit facilitating activities. Similar observation occurred for CD3 as a CF marker. Although the removal of CD3 + cells by negative selection criteria did not eliminate CFs, CFs are shown CD3 + by the sorting cell and to add previous experiments. The cell sorter is much more sensitive and accurate for identifying low density antigens on cell surfaces.

In addition, it was also originally thought that the population facilitating cells was Class II bright. This determination was made by inference based on studies about antibody depletion in anti-Class II. In subsequent positive selections added to the previous experiments, it was shown that the facilitating cells were not in the bright Class II fraction, but expressed the Class II molecules in the range of weak to intermediate level in staining. This was determined by antibody staining and flow cytometry in which the three levels of staining intensity are distinguished by comparison with other cell types such as controls. For example, class II cells - bright spots of Class II + antigens were used as background and bright spots, presenting such cells as B cells and dendritic cells were considered Class II - bright. On the other hand, simultaneous morphological studies described in this section (section 7, infra ) with electron microscopy have identified the bright Class II fraction as lymphocytes. Thus, CFs are Class II positive, but not Class II bright, compared to B cells.

The invention is analyzed in more detail in the following sections, for description purposes only and not to limitation mode For clarity of debate, the specific procedures and methods described in this Document are an example using a murine model; they are merely illustrative for the practice of the invention. Analogues procedures and techniques are equally applicable to all mammal species, including humans, in terms of the use of CF used as a donor and a human recipient receiving such cells in the transplant. Therefore, human CF, with a similar phenotype and function it can be used in conditions described in this document. In addition, CF of animals does not human can also be used to improve the grafting of Xenogenic cells in human patients.

5.1. Characterization of facilitating cells

The mixed chimerism model, in which syngeneic (host), more or allogeneic or xenogenic (donor) of bone marrow are co-administered following Total lethal body irradiation has allowed the identification of the facilitator cells. Bone Marrow Donor Treatment inoculum to remove, or to select and then add-back various cellular subgroups for the cord depleted bone of T cells using RAMB or THY1 or antibodies monoclonal for various CD markers, has revealed a dramatic influence on bone marrow grafting allogeneic and the general level of mixed allogeneic chimerism. Yes TCD of both the sygeneic and allogeneic components of the inoculum bone marrow mixture is carried out, a mixture of Multilineage lymphohematopoietic chimerism occurs. There is evidence that both the genetically modified and the allogeneic stem cells co-injetam, since a mixture of red blood cells from donor and host, platelets, T-cells, B cells, myeloid lineage cells, and NK cells are detectable Each lineage is regulated independently since the level of lymphoid chimerism is not identical to that of Other lineages. However, the percentage of allogeneic chimerism of each lineage remains remarkably stable, with little fluctuation, for the life of the recipient, up to about 12 months.

When the allogeneic component of the inoculum bone marrow is depleted of cells expressing THY1, mixed chimerism is observed. In contrast, if untreated allogeneic bone marrow is administered, the facilitation of allogeneic stem cell grafting resulted, and 100% allogeneic chimerism occurs. This effect is very potent since dose adjustment studies show that the graft facilitation effect is obtained when allogeneic TCDs from bone marrow cells do not graft; resulting in restocking exclusively. Similar facilitation of allogeneic stem cell attachment occurs if CD4 +, NK cells, mature monocytes and macrophages, or B cells are removed. Thus, the mixed chimerism model offers an in vivo model for the identification and characterization of cells capable of facilitating grafting activities.

The studies described in this document demonstrate that facilitator cells are not given stem cells that (1) anti-RAMB treatment eliminates the facilitating effect of allogeneic bone marrow grafting in the rodents, in which grafting occurs more easily than in humans, but mixed chimerism results (in contrast - with 100% allogeneic grafting), and (2) the THY1.2 depletion of mouse allogeneic bone marrow it also eliminates the facilitating effect, but the balance of Syngeneic: allogeneic grafting in the form of mixed chimerism It is slightly larger than a subsequent RAMB treatment. In spite of that some batches of RAMB are well characterized to eliminate bone marrow stem cells, this effect is excluded in synergic reconstitution studies (A → A) before its use in other experiments, since the elimination of all bone marrow stem cells would result in death for Graft failure.

One possible explanation is that the depletion of THY1.2, or the depletion of bone marrow RAMB, leads to the selective depletion of bone marrow progenitor cells. However, when a number range of TCD versus untreated bone marrow gene cells was administered for lethal irradiation to mice, survival curves are similar for both groups, indicating that both bone marrow preparations have a similar ability to reconstitution. In this case of synergic reconstitution, "facilitating the cell" which is relatively radioresistant, exists endogenously in the recipient mouse. Therefore, stem cell depletion after antibody treatment is unlikely to reduce the levels of chimerism seen in TCD receptors versus untreated allogeneic bone marrow.

In addition, hematopoietic stem cells and CF described in this section have a different profile in cell surface expression marker. Patents of United States No. 5061620 intends to characterize the stem cells of the bone marrow as for most cases CD34 +, CD3-, CD7-, CD8-, CD10-, CD14-, CD15-, CD19-, CD20-, CD33-, and THY1 +. By On the other hand, it is believed that the true hematopoietic stem cells  They are in Class II-. The THY1 marker is present in mouse T-cells, NK cells, and some cells myeloids, while T-cells are absent in mature in the rat and human. When the purified cells are transplanted into a genetically identical receptor, the grafting usually occurs. However, these cells highly purified do not graft into allogeneic receptors or genetically different xenogenic.

The studies described in this document show that pluripotent bone marrow stem cells do not They are Class II positive. The use of two different approaches to remove positive Class II cells (flow cytometry with negative selection and antibodies plus complementary treatment), the facilitating effect for stem cell grafting allogeneic is eliminated, discarding the complete grafting allogeneic leads to multilineage mixed chimerism. If the stem cells had been removed from these decreases, the exclusively synthetic restocking should have occurred.

Although the bone marrow stem cell fraction described in US Patent No. 5061620 is also THY1 positive, the distinction between low positive THY1 versus bright positive THY1 is critical. The reference to this difference allows an approach to enrich the stem cells, since the level of expression of THY1 antigens in stem cells was not appreciated by those skilled in the art until recently (Spangrude et al ., 1988, Science 241: 58 ) and was a critical factor to allow the separation of stem cells progeny (more mature) against less differentiated cells. (See also Spangrude, 1989, Immunology Today 10: 344). In addition, a recent report shows that there is no THY1 expression on stem cells in mice that possess the THY1.2 allele (Spangrude and Brooks, 1992, Blood 80: 1957).

In contrast, the purification of - cells facilitators are based on enrichment for the fraction of Class II positive cells. Moreover, these cells by themselves alone in the absence of stem cells do not reconstitute a lethal syngeneic irradiation (A →) in the receptor, while that only 50-100 of the stem cells are are enough to rescue lethal radiation in the mouse.

Detailed analysis of the human bone marrow, following the same procedures reveal a population of Class II + cells with similar front and lateral scattering in flow cytometry that are CD34-. It is believed that this represents the population of human facilitating cells. Like cells corresponding to rodents, human cells have a negative effect for B cells (CD19, CD20), the monocytes / macrophages (CD14), and T-cells (CD4-TCR, CD3) markers. A monoclonal antibody equivalent to anti-CD34 does not exist for cells mother of rodents so that the comparison with staining of CD34 against the alleged facilitation of cell population no It can be done.

How CFs are a novel cell population it is possible that CFs express other markers that have not yet been identified. If so, the previous failure to identify of these unique molecules could be due to their decrease or the lack of expression in the other types of hematopoietic cells. Therefore, CFs can be used to generate antibodies against antigens on its cell surface in order to Identify and characterize such unknown markers. These antibodies can be useful in further purification and characterization of these cells.

Also in the scope of the invention is the production of polyclonal and monoclonal antibodies that recognize new antigenic markers expressed by CF, especially humans and rodents of origin. Several procedures known in the state of the art can be used for the production of antibodies to these cells after they have been isolated. For the production of antibodies, several host animals can be vaccinated by injection with viable, purified or partially purified CF fixed membrane cells, including, but not limited to, those of rabbits, hamsters, mice, rats, etc. Several adjuvants may be used to increase the immune response, depending on the host species, including but not limited to Freund (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lisolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially human useful adjuvants such as BCG (Calmette-Guerin bacillus) and Corynebacterium parvum .

Monoclonal antibodies for novel antigens on CF may be prepared by using any technique that provides for the production of antibody molecules through continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the most recent human B-cell hybridoma technique (Kosbor et al ., 1983, Immunology Today 4 : 72; Cote et al ., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030) and the EBV-hybridoma technique (Cole et al ., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R .Liss, Inc., Pp. 77-96).

Singeneic, allogeneic and xenogenic guests can be immunized with CF that can be prepared viable, or in fixed form, or as fragments extracted from membrane. Monoclonal antibodies can be examined differently. selectively linked to CF, but not to mature macrophages, granulocytes, dendritic cells, T and B cells and the mother cells.

Fragments of antibodies containing side molecule binding can be generated by techniques known. For example, those fragments include, but are not limited to. a: F (ab ') 2 fragments that can be produced by Pepsin digestion of antibody molecule and fragments Fab, which can be generated by the reduction of bridges of disulfide of the F (ab ') 2 fragments.

The activity of the CF in the increase of donor cell grafting also suggests a mechanism which involves the interaction of cells and / or cytokine production. In order to identify possible new cytokines produced by CF, long-term CF cultures can be established or Continuous cell lines can be generated by the transformation of CF into tumor cells using a virus or a chemical Supernatant cultures can be analyzed directly by its application in various cell types used as indicators that are known to respond to certain cytokines in bioassays. Cells can be metabolically labeled and their supernatants subjected to analysis  biochemical. After having identified one of the main proteins by SDS-PAGE and / or biological activity, the protein can be purified by gels SDS-preparations, exchange chromatography ionic, and gels focusing on isoelectric. Purity of Proteins can be verified by SDS-PAGE, quantified by protein assays, confirmed its activities confirmed in bioassays, and use as immunogenes for production of polyclonal and monoclonal antibodies.

The purified proteins can be even more tested in bioassays to stimulate and / or inhibit the proliferation and / or differentiation of a variety of lines indicator cells of various types of tissues. Protein Radiolabels can also be used to identify your receiving cell surface by methods such as labeling of affinity Antibodies specific to cytokines can be used to identify and quantify the forms of membranes and secreted forms of cytokines, to study the biosynthetic pathways, to purify the affinity of proteins immuno temperate penetrate expression libraries for the molecular cloning of the coding sequences.

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5.2. Isolation of facilitating cells

The present invention provides methods of enrichment and / or purification of CF from bone marrow or other physiological sources of hematopoietic cells. The activity of the CF allows its use in a relatively small number when it is enriched from its original source, and absolute purity is not necessary. CF can be isolated from any tissue where they reside, using a variety of separation procedures. Section 7, infra , presents variants of methods such as the illustration to isolate CF from the bone marrow. In accordance with this aspect of the invention, CF can be isolated by separations based on the presence or absence of specific markers.

Although bone marrow is preferred, other sources Physiological hematopoietic cells can be used, by example, the spleen, thymus, blood, embryonic yolk sac, or the fetal liver that come from cells not derived from embryos humans. The bone marrow is preferably removed from the femora or warm, but can also be removed from the spine or other bone cavities. The bone marrow can be removed from the bone cavity by various methods well known to subject matter expert, including bone washing with a mixture of physiological media, balanced saline solution, physiological buffer, and other natural factors. Usually, The bone marrow is filtered, centrifuged and resuspended.

Once a source of cells hematopoietic is obtained, hematopoietic CF can be obtained by various methods that use specific antibodies that bind preferably specific markers to select those cells that possess or lack several markers. These techniques may include, for example, flow cytometry using a fluorescence activated cell sorter (FACS) and specific fluorochromes, separations biotin-avidin or biotin-streptavidin using conjugate with biotin for cell surface markers - antibodies specific and avidin or streptavidin linked to solid support such as an affinity column matrix or plastic surfaces, magnetic separation, using antibodies coated with magnetic balls, destructive separations, such as antibodies and complement or of antibodies linked to cytotocins or radioactive isotopes.

Separation through antibodies to specific markers can be by negative procedures or positive selection. In a negative separation, they are used antibodies that are specific for markers present in undesirable cells. Cells bound by an antibody may be removed and the rest of the desired mixture maintained. In the positive separation, specific antibodies for markers  present in the desired cells are used. Bound cells by the antibody they are separated and maintained. It is understood that the positive and negative separations can be used simultaneously and substantially or sequentially. I also know understand that the current invention encompasses any technique of separation that can be isolated by cells based on the phenotype characteristic of the CF, as its disclosure in this document.

So far, the most common technique for antibody-based separation has been the use of cytometry of flow, as per a FACS. Normally, cytometry separation Flow is performed as follows. The suspension mixture of hematopoietic cells is centrifuged and resuspended in the media. Antibodies that conjugate with fluorochrome are added to allow the binding of antibodies in specific markers of cell surface The cell mixture is then washed by one or more centrifugation and resuspension steps. The mixture passes through of a FACS that separates cells based on different fluorescence characteristics FACS systems are available at different levels of performance and capacity, including a multi-color analysis The facilitator cell can be identified by forward dispersion characteristics and laterals that is influenced by size and granularity as well as for positive and / or negative expression of certain markers of cell surface

Other separation techniques, in addition to Flow cytometry can provide faster separations. A  of these is the avidin-biotin method based on the affinity separation of chromatography. Normally, this kind of technique is performed by incubating the bone marrow washed with biotin from antibodies next to specific markers followed by step a through a column of avidin. Interation Biotin-antibody-cell bind to the column through the interaction avidin-biotin, while other cells pass to through the column Finally, linked column cells They may be released by disturbance or other methods. The specificity of the biotin-avidin system is very Suitable for rapid positive separation.

Flow cytometry and techniques biotin-avidin provide very specific means cell separation media. If you wish, a separation can be initiated by less specific techniques that, however, can  eliminate a large proportion of cells "non-facilitators" of the cell source hematopoietic For example, magnetic ball separations can be used to initially eliminate "non-facilitators" differentiated hematopoietic cell populations, including T-cells, B-cells, naturally killer cells (NK), and macrophages (MAC) as well as smaller cell populations including megakaryocytes, mast cells, eosinophils and basophils. It is advisable that, at at least about 70% and usually at least about 80% of the total hematopoietic cells present can be removed.

A preferred initial separation technique is separation by density gradient. In this sense, the bone marrow or other preparations of hematopoietic cell mixtures is centrifuged and the supernatant removed. The cells are resuspended, for example, in RPMI 1640 medium (Gibco) with 10% FCS and placed in a density gradient prepared with, for example, a Ficoll or Percoll or Eurocollins medium. The separation can be carried out by centrifugation or it can be done automatically with, for example, a Cobel & Cel 2991 Separator (Cobev, Lakewood, Colorado). Additional separation procedures may be convenient depending on the origin of the hematopoietic cell mixture and its content. For example, if blood is used as a source of hematopoietic cells, it may be desirable to separate red blood cells before separation of any fraction. On the other hand, the decantation can also be used alone or in combination with all other purification procedures described in this document (Noga et al ., 1990, Prog. Clin. Biol. Res. 333: 345; Noga et al ., 1992, Prog. Clin. Biol. Res. 377: 411).

CF is generally characterized by being α? -TCR-, δ δ-TCR, CD4-, CD5-, CD16-, CD19-, CD20-, CD56-, mature myeloid lineage- (CD14-), class II +, CD45 +, CD45R +, THY1 +, CD8 + and CD3 +. A high concentration of CF can be obtained by the positive separation of a mixture of cells hematopoietic in a fraction contain facilitating cells that It is class II + and THY1 +. The Class II + fraction may be more separated on the basis of intensity spots and Class II Bright population removed.

A high concentration of CF can be obtained by the positive separation of a mixture of hematopoietic cells in a facilitator cell that contains a fraction that is Class II + and CD45R +. A higher concentration of CF can be obtained by separation of a mixture of hematopoietic cells in a fraction which is Class II +, CD45R +, and THY1 +.

As indicated above, the markers specific used to separate cells will depend on the source of the hematopoietic cell mixture. About 1% at 8% of the bone marrow is Class II positive. At least 80% of bone marrow cells are removed by selection negative using the markers described in this section whose facilitator cells do not have. If the source of cells Hematopoietic is the bone marrow, a high concentration of CF is can get for a large number of different sequences of negative selection. An even higher concentration of CF can be obtained by the positive separation of the bone marrow in a fraction that is class II +. Even a higher concentration can obtained by separating this class II + fraction further into a fraction which is CD19-.

Although the separations based on specific markers were already discovered, it will be understood that the current invention encompasses any separation based on the characterization of the CF disclosed in this document that will result to a cellular composition comprising a high concentration of CF, if that separation is a negative separation, a separation positive, or a combination of negative and positive separations, and if the separation uses cell sorting or any other technique, such as antibodies plus treatment complementary, column separations, panning, technology biotin-avidin, density centrifugation gradient, or other techniques known to those skilled in the matter. It is appreciated that the present invention encompasses these separations used in any mammal, including but not limited to humans, primates, baboons, Rats, mice and other rodents.

The origin of the cell mixture hematopoietic will determine the amount of mixture needed to get a sample large enough from CF. The fountain of the hematopoietic cell mixture will also determine the Time needed to obtain a large enough sample. For example, the concentration of CF in the blood is relatively minute and the separation of a purified fraction of the CF from the blood require a lot of blood and a while relatively long to separate compared to, for example, the use of bone marrow as a source of the mixture of hematopoietic cells.

CF accounts for about 0.5% and 8% of the cells found in the sources of physiological hematopoietic cells. Separations such as those disclosed herein may produce cellular compositions composed of a substantially greater number of CF than that found naturally in physiological hematopoietic cell sources. For example, cellular compositions in which at least 30% of the cells are hematopoietic CFs and are characterized as indicated hereinbefore, and cellular compositions in which at least about 95% of the cells are hematopoietic CFs, and which is also characterized as indicated earlier in this document. An appropriate selection of markers can provide a substantially pure population of CF for in vivo use.

5.3. The uses of facilitating cells

The ability of the CF to improve bone marrow donor cell grafting in a recipient allogeneic or xenogenic indicates that it may be useful to facilitate therapy of various protocols that involve procedures of transplant. The formulation of a cellular composition comprising a high concentration of hematopoietic CF offers a solution alternative to GVHD problems and graft failure. On the other hand, the reduced T cells of the bone marrow donors, With CF retention, they can also be used for transplant. The present invention provides the use of the CF in the establishment of an allogeneic chimeric immune system or mixed xenogeneic. In general, the methods of the present invention refer to the administration of compositions Cells that include CF from purified donors in a receptor, together with stem cells from MHC-specific donors and any additional component of the donor's bone marrow that desired, even though T-cells are preferably reduced. If mixed or complete allogeneic or xenogenic chimerism is desired, the synergic or autologous cellular composition, which comprises CF and stem cells, are administered together with the compositions donor cell phones. However, it is not necessary for the CF to be used with other donor cells that are autologous cells or Syngeneic for the guest. Allogeneic or xenogenic CF can be use with bone marrow cells MHC-matching to reconstitute a receptor, without the co-administration of cells from the gene donor or autologous

Studies conducted to demonstrate that the CF of the present invention are not yet T-cells are capable of improving grafting have been performed in both allogeneic and xenogeneic adjustments (see examples 6-10, infra ). It should be noted that allogeneic bone marrow transplantation seems to be the easiest to perform in certain animal models such as mice among the different strains. Therefore, a donor mouse bone marrow cell treated with RAMB or anti-Thy antibodies are - usually still capable of causing some degree of allogeneic host chimerism, although at a lower level of grafting than if reconstitution It is carried out with CF retention. However, xenogenic bone marrow transplantation of rat donor cells in mice is generally more difficult to achieve with donor cells after TCD, as manifested by the death of the receptors as a result of graft failure. The xenogeneic bone marrow transplant results closely resemble the result seen in allogeneic bone marrow transplantation in humans in which TCD of allogeneic donor bone marrow cells results in high mortality due to graft failure. Therefore, the ability of CF to improve xenograft and xenogeneic chimerism in animal models indicates that these cells can be used successfully in allogeneic bone marrow transplantation in humans. For reasons that are unknown, allogeneic bone marrow transplantation in dogs and pigs seems to be the most difficult to achieve.

CFs are able to facilitate grafting of stem cells and other components of the bone marrow that are MHC-specific for CF. It is possible that particular species or of certain species strains individuals possess CF that are also able to facilitate the grafting of stem cells and other marrow components bone that are not MHC-specific, such as traditionally understood, for the facilitator cell. For more comfort, these CFs will be understood as universal CFs. Compositions cellular comprising such cells are also encompassed in The present invention. On the other hand, it is possible that CF and Stem cells do not have to be matched in your MHC completely. There are two subregions within MHC Class I and II genes. For the therefore, the one corresponding to only one of these regions may be enough for CF to improve the grafting of stem cells. Studies aimed at defining such important MHC subregions they were better developed in mice, using various strains of Commercially available MHC recombinant mouse.

In general, the FC purified or partially purified facilitates the grafting of stem cells that are MHC-specific for CF in order to contribute for the longer survival of the chimeric immune system. The stem cells and CF preferably come from a donor or genetically identical donors. However, if the donor is from a species or a strain of a species that has a cell universal facilitator, stem cells do not have to be MHC-specific for the facilitating cell. To the purify the CF separately, either by positive selection, or by the negative selection, or a combination of selections positive and negative and then the administration of al receptor together with MHC-specific stem cells and any additional components of bone marrow donors that desired, the T-cells that cause GVHD can be removed without fear of graft failure. As a result of this, a mixed or totally allogeneic or xenogenic repopulation It can be achieved.

The embodiment of a method of establishing a allogeneic or xenogenic chimeric immune system is composed  substantially by the destruction of the immune system of receiver. This can be done by well known techniques. by experts in the field. These techniques result in a complete and substantial ablation of the bone marrow stem cells Bone of the recipient. However, there may be some stem cells resistant of the receiver, which survive and continue to produce specific immune cells. These techniques include, for example, lethal irradiation in the receptor with levels of Selected radiation, with specific toxins in the receptor, and with the administration of specific monoclonal antibodies together with active toxins or radio-isotopes, or combinations of these techniques.

Bone marrow is harvested from the long bones of donors. For allogeneic chimerism, donor and recipient are of the same species; For xenogenic chimerism, donor and recipient are of different species. A cellular composition comprising a high concentration of CF is separated from cells of other bone marrow donors through methods disclosed in section 5.2, supra . A cellular composition comprising a high concentration of hematopoietic progenitor stem cells is separated from the rest of the donor's bone marrow. The separation of a cellular composition comprising a high concentration of stem cells can be performed by techniques such as those used to purify CF, but based on different markers, especially with CD34 stem cell separation techniques including the methods disclosed in US Pat. No. 5061620 and the separation of the LC Laboratory Cell Separation System, the CD34 kit manufactured by CellPro, Incorporated of Bothell, Washington. The purified donor facilitating cell composition and the purified donor stem cell composition are preferably mixed in any proportion. However, it is not necessary to mix these cellular compositions.

If the facilitator cell is purified through negative selection using any or all markers disclosed in this document not to express itself in the cell facilitator, then the resulting cell composition will contain stem cells, as well as CF and other immature progenitor cells. Antibodies directed to specific markers of T-cells as anti-CD3 and anti-TCR? can be used to specifically eliminate GVHD-producing cells, while preserving hematopoietic facilitators and stem cells without substantial purification. In that case, this cellular composition can take the place of both cellular compositions referred to above that They comprise both purified CF and purified stem cells.

Purified CF from donors and cells Purified donor mother are then administered in the receivers If these cellular compositions are separated, they they are preferably administered at the same time, but you can administer separately within a relatively narrow period of time. The mode of administration is preferably, but not limited to intravenous injection.

Once administered, it is believed that the cells of origin to various sites of hematopoietic cells in the receptor of the organ, including the bone cavity, spleen, fetal liver or of adults, and scam. The cells are seeded in their places. The cells are grafted and start the creation of a system Chimeric immunological. Since non-universal CF must be MHC-specific, as traditionally understood, with the stem cells whose grafting they facilitate, it is it is possible that both stem cells and CF will adhere to sow the right place for grafting.

The level of allograft or xenograft it is a titulavel effect that depends on the relative number of cells allogeneic and xenogeneic and allogeneic cells and of the type and degree of receiver conditioning. The complete allogeneic chimerism or Xenogenic should occur if the CF of the synergic component have reduced by TCD procedures or other techniques, to condition that an allogeneic or xenogenic CF threshold number be managed. A substantially equal level of syngeneic and allogeneic or xenogenic grafting is requested. The amount of several cells that must be administered are calculated for the specific species of the recipient. For example, in rats, the component of the reduced bone marrow cells of T-cell administered is typically about 1 x 107 cells and 5 x 107 cells per receptor. In mice, the component of the reduced bone marrow cells of T-cell administered is typically about 1 x 106 cells and 5 x 106 cells per receptor. In humans, the component of the reduced bone marrow cells of T-cell administered is typically about 1 x 108 cells and 3 x 108 cells per kilogram of body weight of receiver. The case of inter-species grafting, A larger number of cells may be necessary.

In mice, the number of purified CF administered is preferably about 1 x 104 and 4 x 105 per receiver. In rats, the number of purified CF administered is preferably about 1 x 106 and 30 x 106 per receiver. In in humans, the number of purified CF administered is preferably about 1 x 106 and 10 x 106 per kilogram of receiver.

In mice, the number of stem cells administered is preferably between about 100 and 300 stem cells by receiver In rats, the number of stem cells administered is preferably between 600 and 1200 stem cells per receptor. In humans, the number of stem cells administered is preferably about 1 x 105 and 1 x 106 stem cells per receiver. The amount of specific cells used will depend on Many factors, including the health condition of the recipient. In addition, joint administration of cells with different Cytokines can also promote grafting.

In addition, for total body irradiation, a receptor can be conditioned by immunosuppression and citoreduction by the same techniques that are used in the substantial destruction of a recipient's immune system, including, for example, radiation, toxins, bound antibodies to radioactive toxins or isotopes, or some combination of these techniques However, the level or quantity of agents used it is substantially lower when immunosuppression and cyoreduction that when substantially destroying the system immunological For example, the destruction of a substantial form from the rest of the recipient's immune system often implies lethal irradiation of the receiver with 950 RADS (R) irradiation Total body (TBI). This level of radiation is quite constant. regardless of the species of the recipient. Systematic chimerism Xenogenic (rat? mouse) has been achieved with 750 R TBI and allogeneic systematic chimerism (mouse) with 600R TBI. Chimerism was established by typing e by PBL tolerance confirmed by mixed reactions of mixed lymphocytes (MLR) and by the cytotoxic lymphocyte response (CTL).

As indicated above, the disclosed methods can be used for both the establishment of allogeneic and xenogenic chimerism. Xenogenic chimerism can be established when the donor and recipient as mentioned above are of different species. Xenogenic chimerism between rats and mice, between mice and hamsters, and between chimpanzees and baboons has been established. Xenogenic chimerism between humans and other cousins
This is also possible. Xenogenic chimerism between humans and other mammals is equally viable.

It is appreciated that, although the disclosure of methods previously involve a recipient and a donor, this invention encompasses the application of methods such as those disclosed in which the stem cells and purified CF of two donors are rooted are grafted into a single receptor.

It is appreciated that the present invention also provides methods of restoring the hematopoietic system of a recipient for the substantial destruction of the system immunological receptor or immunosuppressing and cytoreducing the recipient's immune system and then administering to the receiver cellular compositions of purified CF synthetic or autologous and stem cells that are MHC-identical to the FC.

The ability to establish allogeneic chimerism or xenogeneic successfully allows the improvement of the transplant survival The present invention provides the transplantation methods of the physiological component of a donor, such as organs, tissues or cells. Examples of successful transplants in and between rats and mice using These methods include, for example, islet cells, skin, heart, liver, thyroid glands, parathyroid glands, adrenal cortex, adrenal medullas, thymus glands. He chimeric receptor immune system is completely tolerant to the donor of organs, tissues or cells, but competently rejects third party grafts. In addition, the bone marrow transplant confers subsequent tolerance to the organ, tissue, or cell grafts that are genetically identical or very adapted to the bone marrow previously grafted.

The organs, tissues, donor cells transplanted competently perform their function in the recipient. For example, transplanted islet cells work so competent and therefore provide an effective treatment for diabetes. In addition, bone marrow transplantation using methods of The present invention can eliminate autoimmune diabetes trait before developing insulin dependence. The success of solid organ transplants between humans and animals may be carried out using methods herein invention involving hematopoietic CF. For example cells Islets of other species can be transplanted into humans to treat diabetes in the human recipient, after diagnosis of the disease or after the onset of dependence on insulin. The main organs of donor animals as, by For example, pigs, cows or fish can solve the current  Donor shortage problem. For example, 50% of patients requiring a heart transplant die before Make a donor available. It has been shown that acceptance permanent grafted endocrine tissue (thyroid, parathyroid, adrenal cortex, adrenal medulla, islets) occurs in Xenogenic chimeras after bone marrow transplantation of a genetically identical donor. Therefore, xenogenic chimerism mixed or total xenogenic chimerism established by the methods of the present invention can be used for the treatment of endocrine disorders, as well as autoimmunity, such as diabetes

The methods of the present invention involve the transplantation of the specific donor physiological component to through the methods known to those skilled in the art and, in relationship with the establishment of an immune system chimeric in the recipient using the transplant donor as the donor of the purified facilitator cell composition of donor and donor stem cell composition. A Chimeric immune system mixing is preferable. The method of establish a mixed chimeric immune system can be carried carried out before, during or after the transplant, but it is performed preference before transplantation, especially considering that immunosuppression and cytoreduction or immunodestruction is necessary in the chimeric methods disclosed in this document. The Disclosure of methods allows both allotransplantation and xenotransplantation. Because the disclosed methods provide donor-specific immunotolerance, many procedures previously necessary to resist rejection of Donor organs, tissues, or cells are unnecessary. By example, live bone and cartilage can be transplanted through of the method disclosed in this document.

Cell culture technology can provide easy access to the supply of CF, stem cells and physiological components of genetically linked donors. For example, bone marrow cells enriched by the facilitator cell can be propagated through in vitro cultures and / or stored for future transplantation. Cellular material from the same donor can also be stored for future use as grafts.

Beyond transplantation, the ability to establish a successful allogeneic chimeric hematopoietic system or xenogenic or to restore a hematopoietic system or autologous can provide the cure for various other diseases or disorders that are not commonly treated by the bone marrow transplant due to morbidity and mortality associated with GHVD. Autoimmune diseases involve attack of a organ or tissue of the immune system itself. In this disease, the immune system recognizes organs or tissues as strangers. However, when a chimeric immune system is established, the organ relearns what is strange and what is of itself. The establishment of a chimeric immune system as disclosed you can simply stop the autoimmune attack that It causes the disease. On the other hand, the autoimmune attack can be arrested for the restoration of the victim's immune system after immunosuppression and cytoreduction or after immunodestruction with synthetic cell compositions or autologous, as described earlier in this document. Autoimmune diseases that can be treated with this method include, for example, type I diabetes, lupus erythematosus systemic, multiple sclerosis, rheumatoid arthritis, psoriasis, colitis, and even Alzheimer's disease. The use of the CF, in addition to stem cells can significantly expand the scope of - diseases that can be treated with bone marrow transplant.

Because a chimeric immune system includes the hematopoietic cells of the immune system of the donor, deficiencies in the immune system of the receptors can be relieved by a non-deficient immune system of donor. Hemoglobinopathies such as sickle cell anemia, spherocytosis or thalassemia and metabolic disorders such as Hunter's disease, Hurlers' disease, and defects enzymatic, which all derive from deficiencies in the victim's hematopoietic system, can be cured by the establishment of a chimeric immune system in the victim using purified hematopoietic CF from donor and from Donor stem cells from a normal donor. The immune system chimeric should preferably be at least 10% of the donor of origin (allogeneic or xenogeneic).

Ability to establish chimerism Well-succeeded xenogeneic can provide methods of treating or of prevent disease states mediated by pathogens, including viral diseases in which the specific species resistance develop a role For example, AIDS is caused by the infection of the lymphohematopoietic system by a retrovirus (HIV). The virus first contaminates the CD4 + t antigen cells present the cells produced by the stem cells of the bone marrow. Some animals, such as, for example, baboons, they have immunity or resistance native to AIDS. Establishing a xenogenic immune system in a human receptor, with a baboon or an animal resistant to AIDS and / or immune as a donor, the system hematopoietic human receptor can acquire resistance to AIDS and / or immunity of the donor animal. Other states of disease negotiated by pathogen can be cured or prevented by such method using immune or pathogen resistant animals particular that causes the disease. Some examples include the hepatitis A, B, C, and not hepatitis A, B, C. Since the cell facilitator develops a greater role allowing grafting of stem cells through a disparity of species, this approach will trust the presence of the cell facilitator in bone marrow inoculation.

The removal of the facilitating cell was shown to substantially harm or graft through different species. However, when not approximation Preferred, untreated xenogeneic bone marrow will be grafted if the Enough cells were administered. Cells derived from Bone marrow could be used in this case to treat or prevent AIDS with or without enrichment for the facilitating cell. The previous studies showed that GVHD could occur through of a species barrier. Consequently, the approximation preferred would be to establish the xenogenic chimeric immune system using the cellular compositions comprising the donor purified from CF by methods disclosed herein or the compositions depleted of T cells.

In addition, some animals, such as, by For example, babunos and other non-human primates possess immunity or native hepatitis resistance. Transplanting a liver from a baboon or another hepatitis-resistant animal in a victim of hepatitis using a method of the present invention, wherein a Xenogenic chimeric immune system is established in the victim which uses the purified CF donor more stem cells, the donor liver will not be a risk for hepatitis, and the recipient will be graft tolerant, of that mode eliminating the requirement for immunosuppressive agents not specific. The unmodified bone marrow or the purified stem cells may suffice as soon as the liver can serve as a hematopoietic tissue and can contain CF that will promote stem cell grafting of same donor.

The establishment of a mixed chimeric imunological system has also been proven to be effective protective against cancer. Sykes et al ., 1990 Proc. Natl Acad. Sci., USA, 87: 5633-5637). Although the mechanism is not known, it may be due to the multiplication of the specificity of tumor immune cells from the combination of donor and recipient cells of the immune system.

Normally, mixed chimerism is preferred. However, entirely allogeneic or entirely chimerism Xenogenic may be preferable in certain cases. For example, the The present invention provides a method of treating the leukemia or other malignant tumors of the system lympho-hematopoietic substantially comprising the destruction of the victim's immune system and the establishment of an entirely allogeneic chimeric immune system from of the methods described in this document. Since the own The victim's immune system is cancerous, it is preferable to totally replace allogeneic syngeneic cells with cells of a non-cancerous donor. In this case, stem cells purified autologous and the CF can be used with the purpose of totally eliminating cancer cells in the donor preparation, especially if high doses of chemotherapy or radiation is used for endogenous ablation of CF.

The present invention also provides a method of resisting the physiological effects of aging. Current research indicates that aging is related to hormonal changes, such as the decreased growth hormone These changes can result in a physiological decrease and / or protection physicochemical, such as protection against free radicals. The use of methods herein invention, transplantation of the pituitary, pituitary and hypothalamus, or other endocrine tissues can provide levels of Renewed hormone

The present invention also provides methods of practicing gene therapy. Recently it has proved that sometimes same autologous cells that have been genetically modified can be rejected by the addressee. Using methods of the present invention, a chimeric immune system can be established in a receptor using hematopoietic cells that have been modified genetically in the same way as the genetic modification of other cells that were transplanted into it. This will make the recipient of genetically modified cells be tolerant, whether autologous, syngeneic, allogeneic or xenogeneic.

It is appreciated that the present invention presents cellular compositions comprising cellular compositions purified of depleted CF of T cells with retention of CF and of stem cells, CF purification methods, methods of establishing allogeneic chimeric immune systems or Full, total or mixed xenogenic, methods of restoring a syngeneic immune system, and the methods of using CF compositions to treat or prevent diseases, disorders or specific conditions. It is also appreciated that this invention presents the methods of treatment or prevention of certain disease pathogens mediated by xenogeneic cells administered that have not been purified for the cell facilitator

Whereas the incorporations Particular of the invention has been described above, with illustration purposes, it would be evident to experts in the matter that numerous variations of the details can be made without departing from the invention, as defined in the annexed claims.

6. Example: Elimination of facilitating cells reduces the grafting of marrow cells for transplantation allogeneic 6.1. Materials and methods 6.1.1. Chimeras mixture preparation allogeneic

In order to prepare mixed chimeras, the marrow Bone of the long bones of syngeneic mice and mice allogeneic were harvested. The mice were sacrificed with CO2 narcosis, made with 70% alcohol, and long bone posterior (femora and tibia) removed. The cord was emptied of the bones using medium 199 (Gibco Laboratories Life Technology, Inc, Grand Island, New York), supplemented with 50 Pl / ml gentamicin using a 22 gauge needle. The mixture medium (MEM) was used to mechanically resuspend the bone marrow by gentle aspiration through a gauge path of needle 18 and the suspension filtered through mesh gauze sterile nylon The cells were then piloted at 1000 rpm for 10 minutes, resuspended in the MEM, and counted. In the standard allogeneic reconstitution, RAMB was used to T cell depletion (1:40 or adequate dilution at 10 8 cells / ml at 4 ° C for 30 minutes). The cells were washed in the MEM, spin at 1000 rpm for 10 minutes and resuspended in rabbit complement at 37 ° C for 30 minutes (Gibco Life Technology Laboratories, Inc, Grand Island, New York). The cells were washed twice, counted and resuspended in the MEM at the appropriate concentration to allow the injection of 1 ml of Total volume per animal. Within 4-6 hours after irradiation of the recipient animals, the cells were injected through the lateral veins of the tail using a needle gauge 27.

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6.1.2. Animals

Male BALB / c mice six to eight weeks old C57BL / 10SnJ (B10), B10.BR/SgSn (B10.BR), were purchased from the Jackson Laboratory (Bar Harbor, Maine). Rats Fischer 344 (F344), ACI and Wistar Furth (WF) male four to eight weeks old were purchased from Harlan Sprague Dawley, Inc (Indianapolis, Indiana). The animals were housed in a certain installation free of pathogens in the Biomedical Science Tower of the University of Pittsburgh.

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6.1.3. The depletion of the cell subgroups of the bone marrow

When the depletions of the subgroups cell phones were carried out, the bone marrow has been harvested from Similarly. The treatment was carried out using anti-CD4 (L3T4, IgG2b, ATCC or RL1 / 72, IgM), anti-CD8 (LYT2, IgM, ATCC), anti-Thyl. 2 (20-20-5 IgM; ATCC), anti-Mac-1 (IgG2b; ATCC), or anti-Class II IAk (IgM; ATCC), plus rabbit complement (C '), prepared from New White rabbits Zeeland bred at rest and previously controlled in the laboratory. The incubation was at 37 ° C for 45 minutes for antibody treatment followed by washing and 37 ° C for 30 minutes with C 'deal, and washed twice. The rest of the cells were often depleted for a second round with antibodies and complement before use.

Due to anti-NK1.1 anti-antibodies do not fix C ', depletion of NK cells were performed through negative cytometry selection flow. Bone marrow has been harvested in sterile form usual and staining with monoclonal antibodies anti-NK1.1 Hanks performs in saline solution buffered to which more 2% CFS gentamicin was added. The fraction of cells that were not stained with NK1.1 antibodies were collected and used as an NK-negative cell population.

(RAMB) was an anti-soro polyclonal prepared by immunizing rabbits with the brain mouse homogenate. RAMB has been frequently used in the last decades as an agent of cell depletion T.

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6.1.4. Chimeras characterization by cytometry of flow

The receptors were characterized for graft with syngeneic, xenogenic donor lymphoid elements, allogeneic, syngeneic and xenogeneic, or syngeneic and allogeneic using flow cytometry to determine the percentage of peripheral blood leukocytes (PBL) having MHC Class I (H-2b or H-2k) and Class I [RTI] rat anti-F344 [RtIA] l], WF [RtIAu], or the ACI [RtIAa] surface markers. Simply put, the peripheral blood in heparinized plastic serum bottles. After homogenization of the mixture, the suspension was layers more than 1.5 ml of average room temperature separation of lymphocytes (LSM) (Organon Technique, Kensington, Maryland) and se centrifuge at 20 ° C at 1700 rpm for 30 minutes. The layer of lymphocytes was aspirated from the saline-LSM interface and washed with medium. The red blood cells are ACK-zadas (ammonium chloride and potassium carbonate lysis buffer) and the remaining cells stained with monoclonal antibodies appropriate (mAbs) for 30 minutes at 4 ° C and counter stained with sandwich when necessary.

Thymic lymphoid cell analysis and splenic were performed using a cell sorter fluorescence activated (FACS) (FACS II Becton Dickinson and Conpany, Mountain View, California). Monoclonal antibodies anti-WF and Anti-F344-Biotin originate from rats and were used for Class I staining of the cells of rat. Anti-H2 b MAB (28-8-6S) (IgG2a; HB31; American Type Culture Collection, Rockville, Maryland) was used to Class I staining. Anti-CD4-PE mouse, anti-THY1.2 PE, anti-CD8 mouse-FITC (Becton Dickinson and Conpany), anti-TCR - FITC, anti-TCR-, anti-B220 (anti-B cells) and anti-Class II (IAk or CEA) (Pharmangen, San Diego, California) were used for cell staining of the subset.

The data is shown as histograms of cell frequencies in which I record the intensity of fluorescence is shown on the horizontal axis and the relative number of cells in the vertical axis. The percentage of cells considered positive after staining with the MAB has been calculated using a cut line for positivity determined from the fluorescence control profiles of Negative and positive population control (10 B F344 mouse and rat). In addition, the relative size and granularity of the cells were determined by flow cytometry using forward and lateral dispersion. Lymphocytes and other cells with lower size and less granularity resided in a characteristic area, while the largest and most granular cells, such as macrophages and granulocytes resided in another.

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6.2. Results

The experiments described in the following sections will use a mixed chimeric model in which the recipient animals were lethally irradiated and transplanted with different doses and subsets of allogeneic donor cells with or without the co-administration of syngeneic cells. He percentage of allogeneic or xenogenic chimerism, that is, the level of mixed chimerism that was used as an efficiency reading of the donor cell graft.

It should be noted that allogeneic bone marrow grafting between different strains of mice generally occurs with a relatively high frequency, that is, TCD does not fully abrogate grafting. However, xenogeneic grafting is much more difficult to achieve, that is, xenogeneic TCD of donor cells, usually causes the death of recipients due to graft failure. In fact, the results in xenogeneic grafts performed in animal models much more closely resemble results of allogeneic bone marrow transplantation in humans in that TCD of allogeneic bone marrow cells from humans normally leads to a high incidence of mortality. Thus, the data presented in this section, in examples 7 and 8, infra , in which allogeneic bone marrow cells were transplanted were mainly used to demonstrate the activity of CF through the measurement of an increase of chimerism to a lesser degree of chimerism achieved by TCD cell donors. However, Example no. 9, the results of xenogenic grafting transplants are presented below, where it was evaluated in relation to the death of recipients receiving TCD xenogeneic cells from donors. Reconstitution of lethally irradiated receptors with syngeneic TCD (of the host type), plus allogeneic TCD (donor type) of bone marrow (A + B, A) resulted in mixed lymphohematopoietic chimerism Table 1; Group A). When only the inoculum component of the syngeneic bone marrow is TCD with RAMB, a completely allogeneic graft results (Table 1; Group B). Therefore, the syngeneic bone marrow stem cell was not eliminated through TCD, but the cell that facilitates its grafting was.

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TABLE 1

one

TCD is almost certainly not an effect of stem cell depletion, since in the study of syngeneic reconstitution, titration of the number of cells for achieve grafting showed similar survival curves if do not receive treatment or TCD bone marrow was administered. Results similar were obtained when treated RAMB marrow was administered or when monoclonal antibody treatment was used anti-THY-1 plus C '. In others studies using the model of the allogeneic chimera mixture, it demonstrated that the removal of CD4 +, CD8 +, CD4 + plus CD8 + cells, and MAC-1 + by means of monoclonal antibodies plus C ' does not eliminate allogeneic grafting, that is, 100% result allogeneic chimerism (Table 1; Groups C through F). This finding is clinically especially important because cells that express these markers seem to produce GVHD in beings humans, mice and rats. CD4 + cells, CD8 + cells, B cells and to a lesser extent NK cells have been implicated in GVHD Lethal and non-lethal. The elimination of these subgroups, so Therefore, it would eliminate GVHD, but not the facilitator cell.

The adequacy of exhaustion of the models of the mixed allogeneic chimera of Table 1 was confirmed by flow cytometry using a non-reaction crossed with monoclonal antibodies or a saturation technique antibody sandwich (if a second antibody is not available not blocking against the same antigen). Animal receptors were typed at the levels of allogeneic and syngeneic chimerism using anti-Class I monoclonal antibodies (H-2K and H-2b) and PBL at 6 weeks after reconstitution. Some animals were rewritten at 2, 4 and 6 months to follow the chimerism kinetics.

The treatment of allogeneic bone marrow inoculum with anti-THY1.2 plus C to kill cells THY1.2 + result in a reduction of the facilitating effect, represented by the mixture the allogeneic grafting site full. The effect with anti-THY1.2 was not so dramatic as with RAMB that could be the result of the inability of anti-THY1.2 to eliminate by Complete all Thy1.2 + cells. This treatment did not eliminate the allogeneic stem cell, since some allogeneic grafts It was observed; so you must have eliminated the effect facilitator that occurred when the untreated cord was administered (Table 2). Complementary checks were performed to each experiment and the results were similar to those of marrow untreated bone.

TABLE 2

2

In new studies to characterize the potency of this facilitating effect and make a stimulation of the number of cells necessary for the effect, cell titration of Donors were performed to determine the dose at which the effect facilitator was removed, as demonstrated by chimerism mixing or restocking (see Table 3A). While the allogeneic bone marrow cell grafting did not occur at all when 5 x 106 RAMB of allogeneic bone marrow cells treated were administered (Table 3A; Groups 1-4), 100% of the animals were completely chimeric when 5 x 106 cells without treatment (Table 3A; Groups 15 and 16) or reduced of CD4 (Table 3A Groups 6-8) or reduced CD8 (Table 3A, Groups 9-11), or reduced CD4 + CD8 (Table 3A, Groups 12-14) of the allogeneic bone marrow They were administered. Similar results occurred when MAC-1 + cells (Table 3A Group 17) or B220 + cells (Table 3, Group 19) were withdrawn. Table 3B presents data additional that will be cumulative to those presented in Table 3A. These data further support the conclusion that the cell facilitator is a cell separate from stem cells pluripotent hematopoietic since the removal of CD4 + cells and CD8 + would enrich for stem cells, however the complete allogeneic chimerism began to disappear when <5 x 106 allogeneic cells were administered.

TABLE 3A Titration of the Number of Cells in the Component Allogeneic Bone Marrow Grafting: Effect of the composition in Chimerism Level

3

TABLE 3B Negative subset selection effect allogeneic cells (B10.BR) in grafting facilitation of the allogeneic stem cell (5 x 10 6 RAKB B10 + 15 x 10 6 treated B10.BR \ Bigh)

4

5

Natural killers (NK), the cells have notified to exert an influence on the grafting of allogeneic bone marrow grafts. These cells express markers THY1 and NK 1.1 in the mouse, NKRP1 in both mice and rats, and CD16 and CD56 in humans. Since antibody anti-NK 1.1 does not fix complement, NK 1.1+ cells were negatively selected by flow cytometry and the allogeneic rest-NK1.1 inoculum bone marrow used as a cell donor to prepare allogeneic chimeras mixed. In 4 out of 4 recipients tested they received mixed 5 x 106 RAMB treated B10 and 5 x 106 NK 1.1 sold out B10.BR bone marrow, complete allogeneic reconstitution was observed (100% B10.BR), which shows that the facilitator cells were not a NK cell

Similar reduced antibody of plus C was carried out using anti-Class II monoclonal antibody (I-Ak) plus complementary treatment. However, it is well known that the death of Class II by this approach is not as effective as anti-Class I, or antibody negotiated antibody subset targeted subset. Table 4 shows the results of one of the three experiments performed. These data indicate that Class II depleted using mAB plus C 'eliminated the allogeneic facilitating effect in a manner similar to RAMB. However, since the treatment of mAB and C 'in this case is not the optimal approach, negative selection experiments using flow cytometry and monoclonal antibodies directly labeled for positive cell classification was performed as described in section 7. , infra .

It has been widely observed that cells that express a cell surface marker at a low density are less likely to be eliminated by targeted antibodies against this marker, such as lysis of complement-mediated. This may explain the data. relating to the expression of class II molecules in CF, as well as the disparity in CF expression of CD8 and CD3 when tested by antibody plus complement as opposed to selection positive by cell classification using antibodies. Cell classification is a very precise technique that allows the identification of small populations of cells that express low levels of certain surface markers. It is very probable that most CF express CD8, CD3, and Class II at very high levels low.

TABLE 4

6

These data demonstrate that the facilitator cells are not lysed by antibodies specific for CD4, CD8, a CD4 in tandem and CD8, NK1.1, Mac-1 or B220. Consequently, the negative selection of the cells that possess these markers would remove the cells producing GVHD and enrich for CF. After this procedure, at least eighty percent (80%) of the total cells would be eliminated. Negative selection of cells that possess these markers would be a viable clinical approach to preserve and enrich for CF by eliminating GVHD production cells. Subsequent studies using positive selection show that the CFs are CD8 + (see section 7, infra ). These seemingly contradictory results are probably due to incomplete removal of CD8 + cells in the negative selection method, that is, there are not enough CD8 molecules on the surface of CF for a cytotoxic effect when treated with the antibody and complement. . Thus, it may not be an ideal approach to use the anti-CD8 antibody to remove GVHD production cells in an attempt to preserve CF, unless the antibody is first selected for its activity. In addition, mouse CF was not removed by depletion of anti-CD3 but was shown to be CD3 + by positive cell classification. Mouse CF was not removed by the treatment of anti-CD3 + C 'in the transplantation of the mouse bone marrow? Rat. Thus, the experimental data disclosed in this document show that antibodies specific for the MHC Class II II, CD8 and CD3 antigen may not completely eliminate CF by complement lysis but such markers are in fact present on the surface of CF. when cell classification and previous addition studies are executed.

This observation is particularly important with respect to the expression of CD3 by CF. Since CD3 is a marker that is expressed at high levels by T cells that are preliminary GVHD production cells, it is possible to use anti-CD3 antibodies to selectively deplete T cells, which preserves CF expressing lower levels of CD3. However, in order to use anti-CD3 in this way, it must be pre-selected in vitro and in vivo for this selective activity before use.

7. Example: Addiction of enhancement facilitating cells allogeneic bone marrow graft 7.1. Materials and methods 7.1.1. CF positive selection

The bone marrow was collected from donor mouse B10 and donor B10.BR in a manner previously described, Example 6, supra . B10 bone marrow was depleted of T cells using RAMB and rabbit complement as previously described. The bone marrow of B10.BR was de novo suspended in Hanks Balance Salt Solution (HBSS) with 5 ml Hopes (1 molar) per 500 ml in 70 x 10 6 cells / ml at which FCS 2% It was added. The cells were centrifuged and subsequently the anti-Classe II fluorescein-conjugated monoclonal antibody (FITC) was added at 1:10 dilution in MEM + FCS to treat 50 x 10 6 cells / ml. The cells were incubated for 45 minutes at 4 ° C, then washed twice at 1000 RPM for 5 minutes in HBSS + 2% FCS mixture as a selection medium. The cells were then resuspended in the medium and filtered through nylon mesh and analyzed by the Fluorescence Activated Cell Selector (FACS). The duplo laser system allowed 4 fluorescent parameters and two light scattering parameters to be taxed for each cell analyzed. Residual erythrocytes and double cells and debris were excluded by light scattering and staining of propidium iodide. The compensation for spatial overlays of fluorescein and phycoerythrin, and of fluorescein and propidium iodide was adjusted.

To classify the cell, you sample them stained were kept at 4 ° C throughout the entire procedure of classification. The drops classified were collected in MEM with FCS 10%. After isolation of a cell population by FACS, the sample was diluted 1: 1 in MEM, centrifuged at 1000 RPM for 10 minutes, the decanted supernatant and the cell ball resuspended in 0.5 ml of MEM. The suspension was counted and the concentration was adjusted for injection intravenously in lethally irradiated receptors. In these studies, irradiated B10 mice received 5 x 10 6 marrow cells B10 bone treated with RAMB + 5 x10 6 treated marrow cells B10.BR bone treated with RAMB + positive subsets or negatively classified from B10.BR. The degrees were performed to determine the ratio of bone marrow cells syngeneic for allogeneic in that most receptors povoaria as syngeneic or <10% allogeneic. When the relationship of syngeneic bone marrow cells treated with RAMB: allogeneic treated  with RAMB it was 1: 1 (5 x 10 6 B10 treated with RAMB + 5 x 10 6 B10.Br → B.10 treated with RAMB) 57% of receivers repovorated as syngeneic and the general mean for chimerism PBL allogeneic was 17%.

7.2. Results

The experiences of the negative selection described in Section 6, supra , demonstrated that (1) the removal of the Class II + population from the inoculation of the allogeneic bone marrow removed the facilitating effect, and that (2) Administration of the Classe II + population alone did not lead to allogeneic bone marrow grafting, indicating that the stem cell is not Classe II +. In contrast, with antibody plus complement depletion of unwanted cell types, in which the purity of a maximum of 70-80% of the facilitating cell plus the fraction of the stem cell can be obtained, or classifier of the cell could be used to select fractions containing purity of approximately 96-99% and cell viability> 95%. Data from the positive selection and addition studies above will show that the facilitating cell was Classe II + but not bright Class II. Simultaneous morphological studies by electron microscopy identified bright Class II cells as lymphocytes, probably mature B cells; while the CF exhibited an original non-lymphoid morphology. (See Fig. 1). Therefore, the brightness of the Classe II can also be used as a more additional negative selection marker.

The allogeneic graft facilitation of the stem cell occurred reliably and reproducibly if donor specific selected cells Classe II dim / intermediate, CD45 +, CD45R +, or CD8 + del portal of intermediate direct diffraction and diffraction on the side Low (lymphoid) were administered in a receptor (Table 5). Without However, Classe II dim / intermediate cells of the profile of direct and lateral diffraction that characterized the portal Myeloid did not facilitate grafting, nor the negative fraction putative Moreover, BALB / c third-party cells (H-2d) various MHCs classified for same putative markers did not facilitate grafting of allogeneic stem cell in 4 of 6 experiments. By other part, CD8 + / CD45R + / TCR? Β-FC mouse isolated H-2k xH-2d F1 were capable of enhancing the bone marrow graft B10 (h-2b) and B10.BR (H-2k) depleted of RAMB in mouse B10 resulting in allogeneic chimerism H-2k 100% indicating that haploidentical CF is that is, half combined is enough to facilitate the grafting of bone marrow stem cells. Therefore, the CF must be genetically combined, but only in part, to the cells from the donor

TABLE 5

7

700

These data indicate that a population of allogeneic bone marrow cell CD8 +, CD45 +, CD45R +, Class
II dim / intermediate, but not bright Class II with the size and granularity characteristics of the "lymphoid portal"; It is responsible for facilitating allogeneic stem cell grafting. This could represent a single cell type or a smaller, more heterogeneous cell population. Although the facilitation effect is not eliminated by the depletion of CD8 positive cells using the antibody plus complement, the use of the same mAb for cell classification and previous addition experiments revealed that the population of cells certainly expressed CD8 more apparently was not lysed by the antibody plus complement treatment. It is well known in the art that depletion of the antibody from the cells requires the presence of a high density of the corresponding antigen on the cell surface and, therefore, cells expressing low levels of the antigen cannot be eliminated by an antibody with efficacy, while the same antibody can be used to ligate and select the same population of cells much more promptly and positively.

In order to obtain additional purity in the population of facilitation cells, two previous experiments of classifying and adding cell were executed combining the putative markers of the cell surface in various combinations As in the previous selection experiments negative, 5 x 10 6 RAMB B10 bone marrow cells plus 5 x 10 6 RAMB B10.BR were infused with the putative population of positively or negatively selected cells (Table 6). Additional controls prepared that received a number combination of B10.BR bone marrow cells treated with RAMB of Reliable and reproducible way did not have a facilitating effect (n = 196). Using this approach, a fraction of the cell of Purity ranging from 87 to 99% was obtained. The fraction of the Facilitation cell resided in fractions CD45R + CD8 +, Class II + CD45 +, CD8 + CD3 + on the "Linfoid" portal. In (E) the Table 6, the cells were stained with anti-CD4-FITC more anti-CD8-FITC, therefore, the Results included CD4 + CD8-, CD4 + CD8 +, and CD4-CD8 + cells. In several classification studies of three cores, the cell facilitator has proven to be CD8 +, CD45R + and α? -TCR-. Just 10,000-50,000 classified cells were sufficient to mediate the effect of facilitation. Moreover, the fraction purified from this phenotype was morphologically similar to the Class II fraction dim / intermediate strips microscopy of Transmission electronics and by imunocitochemical analysis. These cells indicate a unique appearance and contain granules. abundant filled with colony stimulation factor of granulocyte macrophages, IL-3 and IL-4

TABLE 6

8

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The facilitation cell and the cells dendritic share some phenotypic markers, but differ in others. Co-expression of class II and CD45R in the facilitation cell suggests that these cells may represent a subset of lineage type cells dendritic However, dendritic cells exhibit a classical histological morphology of interdigitation processes elongated and of the cell surface phenotype that are clearly different from the CF described in this document. In addition, mature dendritic cells are distinct from CF because they are Class II bright and CD8-. What is more important is that dendritic cells that are potent cells that have antigen They cannot facilitate the grafting of the stem cell. The propagation of dendritic cells from mature bone marrow according with conventional methods (Steinman, 1991, Ann. Rev. Immunol. or 9: 271) did not facilitate bone marrow stem cell grafting in the syngeneic / allogeneic model mixed using 5 x 10 5 or  1 x 10 6 dendritic cells.

Class II cell population dim / intermediate positively classified was analyzed for morphology using transmission electron microscopy (FIG. 1). A very homogeneous population of cells that were approximately 8-10 microns in diameter was present. The cells they had a pericitoplasmic skirt relatively free of granules and a large population of more centrally placed granules and densely granulated. Most dense granules had a Dense core reminiscent of alpha platelet granules. The nucleus lobed was indicative of myeloid lineage, but the granules were the opposite of homogeneously dense granules of neutrophils or paracristalin granules characteristic of Eosinophils The presence of a large number of very dense granules and the horseshoe-shaped core makes it very unlikely that this cell is a T cell or a population of B cell, since the lymphoid cells have a rounded nucleus with cytoplasm sparse granules and a high nuclear ratio: cytoplasmic. Further, the facilitator cell do not resemble dendritic cells bone marrow precursors or mature dendritic cells. The Class II population classified bright exhibited morphology classical population of the mature T / B lymphoid cell that is clearly different from the CF. Since mouse T cells do not express bright Class II, the population of Class II It represents more likely mature B lymphocytes.

In order to demonstrate that the CF of the invention were not stem cells capable of giving rise to hematopoietic cells and that the stem cell graft demanded the presence of CF, the stem cells purified and CF were used for allogeneic reconstitution. In this experiment, the stem cells of B10.BR that they have an SCA 1+ and Lin- phenotype (B220-,?? TCR-, GR-1 -, MAC-1 - and CD8-) were isolated at a purity greater than 95% by the classification of cell. It is believed that these cells are equivalent in function biological to human CD34 + stem cells. 50,000 Sca + trunk cells, Lin- were grafted into allogeneic B10 mice irradiated with or without 50,000 B10.BR CF positively classified for double expression of CD8 and CD45R (?? -TCR-). The board 7 shows that stem cells or CF alone they do not reconstitute the recipient mice, while the combination of CF and stem cells led to allogeneic chimerism. While the stem cells alone grafted into syngeneic receptors due to the presence of endogenous CF, the CF alone did not graft, further confirming that Cf are not stem cells.

TABLE 7

10

In short, using positive selections and previous experiments, CF were characterized as being THY1 +, CD45 +, CD45R +, CD3 + Class II dim / intermediate and CD8 +. These cells are necessary for stem cells graft into allogeneic and xenogeneic receptors. Morphologically, the cells do not resemble lymphocytes or any other type of cell described previously. Thus, the CFs are a population distinct cell that expresses an original combination of markers of leukocyte. It seems that the specific MHC interaction: ligand contributes to the success of allogeneic grafting. On the contrary, the B cells, macrophages / monocytes, NK cells, and cells DC4 + and Class II bright do not exhibit activity facilitator

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8. Example: Donor bone marrow depletion of subsets of specific T cells does not decrease grafting of allogeneic cell 8.1. Results

The results obtained from the studies described in Examples 6 and 7, supra , clearly demonstrate that the CF of the current invention is a cell type other than T cells, although certain markers such as Thy-1, CD3, and CD8 are usually expressed by both cell populations. The recognition that T cells can be selectively and specifically depleted by antibodies against markers only expressed by T cells, and not by CF, indicates that TCD of donor bone marrow cells can be used to eliminate cells producing GVHD without endangering the donor cell graft in bone marrow transplantation, if appropriate T cell specific reagents are used for TCD. In fact, in the light of the present invention, results in the art that show donor cell graft reduction as a result of TCD using RAMB or anti-Thy-1 antibodies can now be interpreted to mean that those Reagents used at the time will deplete T and CF cells.

Table 8 illustrates results of the allogeneic bone marrow transplantation performed in mice.  Bone marrow cells of non-modified allogeneic mice will be presented to graft into allogeneic mice to give rise to chimerism mixed when transferred bluntly with cells from the bone marrow of the syngeneic mouse TCD. Although this approach could establish the donor cell graft, it was not a viable clinical protocol due to the high risk of GVHD. For other part, TCD of mouse donor bone marrow cells with RARB did not cause grafting in allogeneic receptors, presumably due to the simultaneous elimination of both CF and GVHD mediation T cells. However, the most important thing is that when donor cells were depleted with a reagent T cell specific such as an antibody anti-CD3 or anti-α? -TCR graft of donor cells was established. It should be noted that yes either CF can be CD3 +, certain antibodies anti-CD3 may be able to remove so selective T cells without significantly eliminating CF.

On the other hand, it is well known in art that lymphohematopoietic chimerism in general correlates with the donor-specific tolerance of a recipient. When mice reconstituted with allogeneic cells depleted of αCD3 were subsequently implanted with transplants from the heart of the donor mouse strain, they will show retain transplants for more than 3 months. On the other hand, the heart transplants of an intercessor mouse strain irrelevant were rejected by recipients within approximately 10 days Similar results were observed. also for skin grafts where grafts donor-specific were accepted and grafts genetically disparate intercessors rejected.

Together, these results indicate that Donor bone marrow cells can be treated with a T-specific cell reagent to deplete only T cells with CF retention for transplant use of the bone marrow. This approach eliminates most cells. responsible for GVHD, without reducing the ability to hematopoietic stem cells graft a receiver. GVHD production cells can still be eliminated hair cell preparation treatment with antibodies specific for B cells and NK cells. In addition, the grafting of donor bone marrow cells establish chimerism in the receiver, causing a tolerance state donor-specific at the recipient to allow the transplantation of any donor cell, tissue or organ establish long-term or even permanent grafting. Of this mode, the presence of CF in a cell preparation of the donor for bone marrow transplantation can be used as a tolerance agent without the risk of GVHD facilitate solid organ transplantation. It should be noted that tolerance induced is donor specific, therefore not immune would compromise the receiver's ability to mount a response immune to other antigens.

TABLE 8

eleven

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9. Example: Facilitating cells enhance the graft of the xenogeneic bone marrow 9.1. Materials and methods 9.1.1. Xenogeneically reconstituted animals (A + B \ rightarrow A)

In the chimeras mouse + rat \ rightarrow mouse, mice received 5 x 10 6 marrow cells mouse bone depleted of T cell plus 4 x 10 7 cells of the untreated mouse bone marrow unless otherwise specified way. TCD performed with the α? Antibodies anti-TCR and complement, or with contas immunomagnetic coupled to the antibody, achieved results Similar. In the mouse chimaera? Rat, mice received 250 x 10 6 non-mouse bone marrow cells Treated or treated.

Under these conditions it has been shown that the most rat T-lymphocytes in chimeras mouse + rat \ mouse mouse were derived from the bone marrow stem cell precursors of the rat and not containing T-lymphocytes no inoculum of the bone marrow, since the maturation of T cell came from a developed form regulated in the thymus. In addition, most of the T cells in the chimeras mouse + rat → mouse were coming from mouse. The radiation controls were prepared for confirmation of the sufficiency of the radiation.

9.1.2. Human Marrow Collection

The human bone marrow was obtained from the vertebral bodies of corpse donors. The vertebral bodies were transported in nutrient-rich medium ( Ex-Vivo ; Whitacker Conpany) supplemented with 500,000 units of polymyxin, 500,000 units of bacitracin and 10% human serum albumin. The vertebral bodies were separated into four parts each. The whole process was done at room temperature. The soft spongy bone was removed outside using rongeurs and the marrow cells evicted by delicate agitation for a total of 90 minutes. At each 30 minute interval, the supernatant was filtered through a double layer mesh penis (pore size 420 micrans; 180 micrans) and 500 ml of fresh media were added. All fractions were combined, centrifuged at 1000 RPM for 10 minutes, counted, and resuspended at a concentration of 20 x 10 6 cells / ml. With this technique, 40 x 10 9 at 60 x 10 9 cells per 5 vertebral bodies were obtained. The flow cytometry analysis was, therefore, performed as described in example 6, supra , to determine its phenotype.

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9.2. Results

The facilitation cell has an effect similar in bone marrow grafting through the barriers of species, for example, rat \ mouse mouse, mouse \ rightarrow rat. When 4 x 10 6 mouse bone marrow cells and syngeneic mouse cells were transferred in mice after of TCD using rabbit-anti-mouse (RARB) or anti-Thy, lethally receptors irradiated were not reconstituted (Table 9). Also, if the mouse bone marrow cells were transferred to mice lethally irradiated after TCD using RARB or anti-Thy 1.1 in the absence of mouse cells syngeneic as donor cells, 100% mortality of receptors resulted due to failure of radiation rescue - induced aplasia and graft failure (Table 10). This result closely resembles the results of bone marrow transplantation human allogeneic bone, in which TCD of donor cells in general it would not result in any grafting and consequently at a high rate of mortality (up to 70%). On the other hand, if the bone marrow does not treated mouse, or mouse bone marrow depleted of cells CD4 + + CD8 + or CD3 + cells or cells α? -TCR + or cells α? -TCR + more B cells or cells α? -TCR + more NK cells was administered, the graft was achieved and> 90% of the recipients have Survived for more than 180 days. It should be noted that mouse CF can be CD8 +, similar to the phenotype of mouse cells corresponding. Thus, the results observed with the negative selection using anti-CD8 can, once more, be due to incomplete removal. The CF for the Xenograft was?? -TCR-, CD3- and CD4- because the facilitation effect was not removed by depleting these cells using immunomagnetic granules or cytotoxity complement mediated. Again, the mouse CF can also be CD3 + but they were not completely depleted by the antibody.

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TABLE 9

12

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TABLE 10

13

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It was also noted that the animals that show Xenogenic chimerism were tolerant to the giver's cells, as measured by a lack of reactivity in the leukocyte reaction mixed as well as the acceptance of heart, skin and islets Consequently, the retention of CF in the population of donor cell not only enhanced the bone marrow graft bone, but also induced a state of tolerance donor-specific, which makes it possible to perform the subsequent xenogeneic or solid organ cell transplantation or simultaneously of a different species. It is important point out that these tissues work properly in an environment Xenogenic

A technique was developed to isolate large bone marrow cell numbers of the vertebral bodies humans. Bone marrow monoclonal antibody staining was executed using techniques similar to those used to rodents, to identify corresponding populations of cells. The analysis of the direct diffraction profile and for the side of the human bone marrow by FACS will identify the cells similar in the phenotype to CF in the bone marrow of the rodent.

A stain of two colors was made to examine if similar cell populations were Class II bright, intermediate Class II and dim, B cell lineage (LEU 12) negative, several CD45 isoforms and negative T cell marker. In one of these studies, the separation of the density gradient from the bone marrow was used before the spot to enrich Variant density cell populations. It has been shown that human bone marrow contained a population of Class II cells positive, negative B cell lineage marker cells similar to the rodent bone marrow CF. In addition, a population Class II bright, cell B was also considered.

To determine if the fraction of the cell present in the human bone marrow, which shared similarities of cell surface marker with rodent CF, could enhance bone marrow stem cell grafting human, a model for mixed xenogeneic chimeras (mouse + human? mouse) was used. The chimeras were prepared in which syngeneic TCD (mouse B10) more untreated human (80x106 cells) were administered in the receptors conditioned with 950 rads of total body irradiation. In a week after reconstitution two animals were sacrificed and its bone marrow, spleen and thymus tissues analyzed for presence of human cells that load surface markers HLA-DR cell (Class II), CD4, CD8, CD19, and CD14. Evidence (<10%) of mixed human chimerism was present in the bone marrow and chimerism <5% was present in the spleen The animals monitored for four months continued to have low but detectable levels of human cells in the bone marrow that is. The low levels of chimerism observed and the absence of mature human blood cells in mice could be due to the inability of human blood cells to Respond to the cytokines of the host mouse. Therefore, the co-administration of growth factors specific such as interleukin-1 and 3, several colony stimulation factors, factor of stem cell and erythropoietin might be able to support the growth and maturation of human cells in the mice. Alternatively, other animal hosts such as baboons that are phylogenetically closer to humans that rodents were also used to examine the Facilitating function in the bone marrow xenograft Human bone in the presence of putative CF. The studies that involve human bone marrow cell transfer not treated (6 x 10 8 cells / kg) in baboons treated with 2200 Rad radiation had a low but detectable graft level of human cells of lineages mixed in baboons.

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10. Example: Mixed allogeneic chimerism prevents autoimmune diabetes and invert insulites 10.1 Materials and methods 10.1.1 Mouse auto-immune model

Non-obese diabetic mice (NOD) were obtained from Taconic Laboratories and housed in a pathogen-free site at the Pittsburgh Cancer Institute. At animal site, femeous NOD mice developed initial diabetes Acute spontaneously at a rate of 65% in six months, and 80% in eight months of age. All animals tested had insulitis in six weeks old To establish mixed allogeneic chimerism, lethally irradiated NOD mice were transplanted with syngeneic bone marrow cells plus bone marrow cells allogeneic of B10.BR or AKR mice. Immunohistochemical analysis of these animals was performed at specific time points after reconstitution.

10.2. Results

The mixed allogeneic chimerism model described in this document was used to prevent development of diabetes in NOD mice. Such mice grafted with cells from allogeneic bone marrow exhibited mixed allogeneic chimerism up to seven months and the onset of diabetes was prevented in all The animals tested. The immunohistochemical analysis of mice within five months after reconstitution put He stated that the islets were free of insulitis. In In contrast, four of 13 mice reconstituted only with cells of the syngeneic bone marrow developed acute diabetes and all The mice had insulitis. These results suggest that the ability to selectively remove the T cells responsible for GVHD and preserve CF to enhance bone marrow grafting allogeneic may allow the extension of transplantation of the bone marrow to a variety of disease conditions that do not They are currently favorable to this modality due to GVHD. The co-administration of hematopoietic CF cells and stem cells can also allow less aggressive cytoreduction of a receptor to allow graft. The diseases that can be treated by this modality include, but are not limited to, self-immunity, immunodeficiency and viral infection just like AIDS.

The present invention should not be limited scope for exemplified incorporations, which are intended to be used as illustrations of individual aspects of the invention. In fact, several modifications of the invention, in addition of those shown and described in this document will be thundered apparent to those skilled in art by the above description and for the attached drawings.

Claims (61)

1. A cellular composition comprising the hematopoietic mammalian cells, which are depleted of the graft versus host disease producing cells having a phenotype of? TCR + and? TCR +, with the retention of hematopoietic facilitating cells of mammal having a phenotype of CD8 + and?? TCR- as determined by the antibody stain and flow cytometry, whose hematopoietic facilitating cells are capable of facilitating the grafting of bone marrow cells and are partially combined with the cells of the donor and whose hematopoietic facilitating cells are not derived from human embryos. 2. The cellular composition according to the claim 1, wherein hematopoietic facilitating cells Mammalian are CD3 +. 3. The cellular composition according to the claim 1 or 2, wherein the facilitating cells Mammalian hematopoietics are δ TCR-. 4. The cellular composition according to any one of claims 1-3, which comprises at least 30% of hematopoietic facilitating cells of mammalian 5. The cell composition according to any one of claims 1-4, which comprises at least 95% of hematopoietic facilitating cells of mammalian 6. The cell composition according to any one of claims 1-5, wherein mammalian hematopoietic facilitating cells are CD45 +. 7. The cellular composition according to the claim 6, wherein the cells are CD45R +. 8. The cellular composition of the claim 7, in which the cells are Thyl +, CD19- and CD56-. 9. The cell composition according to any one of claims 1-7, wherein mammalian hematopoietic facilitating cells are cells human hematopoietic facilitators. 10. The cellular composition of the claim 9, in which the cells are CD19- and CD56-. 11. The cellular composition of the claim 9, which also includes the CD34 + cells that are histocompatible with hematopoietic facilitating cells. 12. The cellular composition of claim 11, wherein the graft versus host disease production cells still comprise CD19 + and CD56 + cells. 13. A pharmaceutical composition to facilitate CD34 + hematopoietic stem cell graft in a receptor, in which the active ingredient is cells hematopoietic facilitators as defined in the claims  1-12 and hematopoietic facilitating cells are histocompatible with stem cells CD34 +. 14. A pharmaceutical composition for bone marrow transplantation in which the active ingredients they are CD34 + hematopoietic stem cells and cells histocompatible hematopoietic facilitators as defined in any one of claims 1-12. 15. The use of the cellular composition according with any one of claims 1-12 or of the pharmaceutical composition according to claim 13 or 14, in the manufacture of a medicament for partially or completely reconstitute the lymphohematopoietic system of a mammal. 16. The use of a cellular composition according with any one of claims 1-12 or of the pharmaceutical composition according to claim 13 or 14, in the manufacture of a drug to induce tolerance donor-specific in a mammal in order to facilitate long-term grafting of cells, tissues or Donor organs 17. The use according to claim 15, in which the mammal is conditioned by the total irradiation of the body. 18. The use of claim 15, wherein the Mammalian is conditioned by an immunosuppressive agent. 19. The use according to claim 15, in which the mammal is conditioned by a cytoreductive agent. 20. The use of claim 15, wherein the Medication is in an intravenous form. 21. The use according to claim 15, in which the mammal is a human being. 22. The use of claim 15, wherein the mammal suffers from auto-immunity. 23. The use of claim 22, wherein the Self-immunity is diabetes. 24. The use of claim 22, wherein the Autoimmunity is multiple sclerosis. 25. The use of claim 22, wherein the Self-immunity is systemic lupus erythematosus. 26. The use of claim 15, wherein the mammal suffers from immunodeficiency. 27. The use of claim 26, wherein the mammal is contaminated with an immunodeficiency virus human 28. The use of claim 15, wherein the Mammalian is infected with a hepatitis virus. 29. The use of claim 15, wherein the mammal suffers from a hematopoietic malignancy. 30. The use of claim 15, wherein the mammal suffers from anemia. 31. The use of claim 15, wherein the mammal suffers from hemoglobinopathies. 32. The use of claim 15, wherein the mammal suffers from a state of enzyme deficiency. 33. Or use of claim 15 or 16, wherein the mammal is a human being and the cellular composition or the Pharmaceutical composition is obtained from a human being. 34. The use of claim 15 or 16, wherein the mammal is a human being and the cellular composition or the Pharmaceutical composition is obtained from a non-human animal. 35. The use of claim 34, wherein the Non-human animal is baboon. 36. The use of claim 34, wherein the Non-human animal is pig. 37. The use of claim 16, wherein the Donor organ is heart. 38. The use of claim 16, wherein the Donor organ is skin. 39. The use of claim 16, wherein the Donor organ is liver. 40. The use of claim 16, wherein the Donor organ is lung. 41. The use of claim 16, wherein Donor organs are heart and lung. 42. The use of claim 16, wherein the Donor organ is kidney. 43. The use of claim 16, wherein donor tissues are pancreatic islet cells or pancreas whole. 44. The use of claim 16, wherein the Donor organ is an endocrine organ. 45. The use of claim 44, wherein the Endocrine organ is a thyroid gland. 46. The use of claim 44, wherein the Endocrine organ is a parathyroid gland. 47. The use of claim 44, wherein the endocrine organ is a thymus. 48. The use of claim 44, wherein the Endocrine organ is adrenal cortex. 49. The use of claim 44, wherein the Endocrine organ is adrenal medulla. 50. The use of claim 16, wherein the Donor cells are neurons. 51. The use of claim 16, wherein the Donor cells are myocytes. 52. A method to obtain a composition cell according to any one of the claims 1-12, which comprises subjecting a cell mixture hematopoietic to negative selection to remove cells expressing? \ betaTCR + and?? delTCR +, since the hematopoietic cell mixture is not derived from embryos humans. 53. The method according to claim 52, in which the cells are removed by an antibody. 54. The method of claim 52, wherein the antibody is conjugated to a magnetic granule. 55. The method according to claim 52, in which the cellular composition is first separated by the density granule centrifugation to obtain cells in the mononuclear fraction of the cell. 56. The method according to claim 52, in which the cellular composition is still depleted of the cells that express CD19 and CD56. 57. The method according to claim 52, in which the cellular composition comprises the cells CD34 + hematopoietic. 58. The method according to claim 52, in which the cellular composition is derived from the bone marrow that is. 59. The method according to claim 52, in which the cellular composition is derived from the thymus. 60. The method according to claim 52, in which the cellular composition is derived from blood peripheral. 61. The method according to claim 52, in which the cellular composition is derived from fetal liver.